Pflügers Archiv

, Volume 447, Issue 1, pp 97–108 | Cite as

Measurement of rapid changes in cell volume by forward light scattering

  • S. P. Srinivas
  • Joseph A. Bonanno
  • Els Larivière
  • Danny Jans
  • Willy Van Driessche
Instruments and Techniques
First Online:
Received:
Revised:
Accepted:

Abstract

Light scattering is an empirical technique employed to measure rapid changes in cell volume. This study describes a new configuration for the method of light scattering and its corroboration by measurements of cell height (as a measure of cell volume). Corneal endothelial cells cultured on glass cover-slips were mounted in a perfusion chamber on the stage of an inverted microscope. A beam of light was focused on the cells from above the stage at an angle of 40° to the plane of the stage. The scattered light intensity (SLI), captured by the objective and referred to as forward light scatter (FLS), increased and decreased in response to hyposmotic and hyperosmotic shocks, respectively. The rapid increase and decrease in SLI corresponded to cell swelling and shrinkage, respectively. Subsequently, SLI decreased and increased as expected for a regulatory volume decrease (RVD) and increase (RVI), respectively. These data are in agreement with measurements of cell height, demonstrating that the method of light scatter in FLS mode is useful for monitoring rapid changes in cell volume of cultured cells. Changes in SLI caused by gramicidin were consistent with cell volume changes induced by equilibration of NaCl and KCl concentrations across the cell membrane. Similarly, an additional decrease in SLI was recorded during RVD upon increasing K+ conductance by valinomycin. Decreasing K+ conductance of the cell membrane with Ba2+ changed the time course of SLI consistent with the effect of the K+ channel blocker on RVD. Bumetanide and dihydro-ouabain inhibited increases in SLI during RVI. In conclusion, FLS is a valid method for qualitative analysis of cell volume changes with a high time resolution.

Keywords

Light scattering Cell height Regulatory volume decrease Regulatory volume increase Corneal endothelial cells Cell volume 
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Notes

Acknowledgements

Supported by NIH grants E11107 (SP) and EY00834 (JAB) and by grant "Fonds voor Wetenschappelijk Onderzoek Vlaanderen" G.0179.00 (WVD). The authors thank Prof. Bernard Himpens, M.D. and Priya Gomez, Laboratory of Physiology, K. U. Leuven, Campus Gasthuisberg O/N, Leuven (Belgium) for their help in obtaining confocal images shown in Fig. 6.

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Copyright information

© Springer-Verlag  2003

Authors and Affiliations

  • S. P. Srinivas
    • 2
  • Joseph A. Bonanno
    • 2
  • Els Larivière
    • 1
  • Danny Jans
    • 1
  • Willy Van Driessche
    • 1
  1. 1.Laboratory of PhysiologyK. U. Leuven, Campus Gasthuisberg O/NLeuvenBelgium
  2. 2.School of OptometryIndiana UniversityBloomingtonUSA

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